Neonatal exposure to bisphenol A or diethylstilbestrol alters the ovarian follicular dynamics in the lamb

Abstract

We hypothesized that neonatal xenoestrogen exposure affects the ovarian follicular dynamics in lambs. Female lambs were exposed from postnatal day (PND) 1–14 to low doses of diethylstilbestrol (DES) or bisphenol A (BPA). At PND 30, the follicular dynamics and ovarian biomarkers (ERα, ERβ, AR, Ki67, p27) were evaluated. Lambs exposed to DES or BPA showed a decline in the stock of primordial follicles with stimulation of follicular development. BPA reduced ovarian weight and increased the number of multioocyte follicles. BPA promoted proliferation of granulosa/theca cells in antral follicles, and increased both the number of antral atretic follicles and p27 expression. Neonatal exposure to BPA or DES reduced the primordial follicle pool by stimulating their initial recruitment and subsequent follicle development until antral stage. In prepubertal lambs, the accelerated folliculogenesis resulted in increased incidence of atretic follicles. These alterations may affect the ovarian function in the adult.

Introduction

Exposure to endocrine-disrupting compounds (EDCs) during critical periods of development can affect gonad formation and disrupt reproductive functions during adulthood [1]. Exposure of cattle and sheep to estrogenic forage, such as subterranean clover (Trifolium subterraneum), reduces conception rates, increases embryonic loss, and impairs ovarian function [2], leading to substantial reductions in livestock productivity [3], [4].

Both diethylstilbestrol (DES) and bisphenol A (BPA) are considered EDCs and have been extensively studied using different animal models. DES is a synthetic estrogen with a stronger bioactivity than estradiol (E₂) [5]. In mice, postnatal DES exposure results in excessive multioocyte follicles (MOFs) and accelerated follicular development [6], [7]. In the past, DES was widely used in human and veterinary medicine, and significant levels of DES may be present in the environment, mainly related to feed-lot areas [5].

BPA is used in numerous manufactured products. In 2004, the estimated BPA production in the United States was approximately 2.3 billion pounds, most of which was used in polycarbonate plastics and resins [8]. Because BPA has been shown to leach from containers into food and beverage products, and it is one of many contaminants in soil, surface water and sludge sewage. Therefore, BPA should be considered a potential health risk for animals and humans [9]. Many studies have clearly shown that BPA has estrogenic properties. In previous works, using rodent and reptile models, we have shown that early BPA exposure impairs the normal development of different endocrine-related tissues [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. Additionally, in utero exposure to low doses of BPA disrupts early oogenesis in mice [21], and postnatal injections of BPA induce MOFs [22]. In contrast to laboratory animals and wildlife, the potential reproductive effects of EDC exposure in domestic animals have received little attention. In sheep, prenatal exposure to BPA reduces the birth weight of offspring, and the exposed-animals are hypergonadotropic and end their breeding season later during adulthood [23]. However, little is known regarding the effects of neonatal BPA or DES exposure on the developmental programming of the sheep ovary.

Because follicular differentiation gets completed in utero, all follicular classes (primordial to antral) are present in early postnatal lamb. Regardless of this early differentiation, EDC exposure during early postnatal life can have an impact on maintenance of ovarian reserve. In this study, we tested whether neonatal exposure to low doses of BPA or DES adversely affects the prepubertal lamb ovary. Specifically, the ovaries were studied on postnatal day 30 (PND 30) to assess whether exposure to xenoestrogens produces adverse effects on follicular development (follicle dynamics, total reserve of oocytes, induction of MOFs, and atretic follicles). In addition, to investigate the potential signaling pathways underlying these effects we evaluated the expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), androgen receptor (AR), Ki67 (a cellular proliferation marker), cyclin dependent kinase inhibitor 1B (commonly known as p27^kip1 or p27), E₂ and testosterone (T) serum levels. Xenoestrogens can act through different steroid receptors in the target cells, thus the ontogeny of ovarian expression of ERα, ERβ and AR during the period of xenoestrogen exposure (PND1–14) was studied. Spatial and temporal patterns of expression of steroid receptors were described in neonatal ovaries of unexposed animals.